Validation of the applied biosystems MicroSEQReg. real-time PCR system for detection of E. coli O157:H7 in food.

摘要

Modern molecular methods offer the advantages of simplicity and short time-to-results compared to traditional culture methods. A new Real-Time PCR method was developed to detect E. coli O157:H7 in 5 food matrixes. The complete system consists of the MicroSEQReg.E. coli O157:H7 Detection Kit, sample preparation (2 sample preparation methods, the PrepSEQReg. Nucleic Acid Extraction Kit and the PrepSEQ Rapid Spin Sample Preparation Kit, were validated), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinderTM Express software. The test method was compared to the U.S. Department of Agriculture Microbiology Laboratory Guidebook 5.04 reference method for detecting E. coli O157:H7 in 25 g and 375 g beef mince and beef trim, and to the ISO 16654 reference method for detecting E. coli O157:H7 in 25 g spinach, orange juice and apple juice. The MicroSEQ E. coli O157:H7 Detection Kit showed equivalent detection compared to the corresponding reference method based on Mantel-Haenszel Chi-square statistics for all matrixes tested. An independent validation confirmed these findings on beef mince. The MicroSEQ kit detected all 51 E. coli O157:H7 strains tested and showed good discrimination against an exclusivity panel of 30 strains.